Nuclear hormone receptors (NHRs) play key roles in development, homeostasis, and disease (Kliewer, Lehmann et al. 1999; Chawla, Repa et al. 2001; Olefsky 2001). Targeted gene deletion of these receptors in mice has proven their association with different diseases including atherosclerosis, cancer, diabetes, and lipid disorders (Horard and Vanacker 2003; Smith and Muscat 2005; Glass 2006). These findings have opened new strategies for treatment of these diseases, and implicate orphan receptors as important targets for drug discovery. Nuclear receptors (NR) act as ligand-inducible transcription factors that are regulated by binding to small lipophilic molecules such as steroid and thyroid hormones or the active forms of vitamin A (retinoids) and vitamin D (Moras and Gronemeyer 1998; Escriva, Delaunay et al. 2000; Aranda and Pascual 2001; Kumar, Johnson et al. 2004). These molecules play an important role in the embryonic development, growth, differentiation, metabolism, reproduction, homeostasis and morphogenesis of higher organisms and humans. Several members of the nuclear receptor family for which ligands have not been identified are classified as orphan receptors (Blumberg and Evans 1998; Giguere 1999).
The estrogen related receptors (ERRs) were the first orphan NR to be discovered and to date three members have been identified (ERRα, ERRβ and ERRγ). The ERR subfamily is closely related to the estrogen receptors ERα and ERβ. ERRα and ERRβ were first isolated by a low stringency hybridization screen (Giguere, Yang et al. 1988) followed later with the discovery of ERRγ (Hong, Yang et al. 1999). Though sharing structural homology with the estrogen receptors, these receptors do not bind estrogens. Unlike classical estrogen receptors that are ligand activated NR, the ERR's show varying levels of constitutive activity that appears to be tissue selective (Kraus, Ariazi et al. 2002; Horard and Vanacker 2003). The ERRs and ERs share sequence similarity with the highest homology observed in their DNA binding domains. They interact with classical DNA estrogen response elements and half sites (Johnston, Liu et al. 1997; Vanacker, Pettersson et al. 1999). Recent biochemical evidence has shown that the ERRs and ERs share co-regulatory proteins and can functionally interfere with estrogen responsive genes in the breast and bone including pS2, lactoferin, aromatase and osteopontin (Hong, Yang et al. 1999; Vanacker, Pettersson et al. 1999; Zhang and Teng 2000; Giguere 2002; Kraus, Ariazi et al. 2002). ERR's in addition to synergizing or competing with estrogen responsive genes have also been implicated in maintaining energy homeostasis (Kamei, Ohizumi et al. 2003). A recently described ERRα knock-out has reduced adiposity and is resistant to weight gain within 3-5 weeks after feeding a high fat diet (Luo, Sladek et al. 2003). Food consumption and energy expenditure were unaltered. Gene expression profiling of the small intestine and adipose tissues of these knock-out animals show alterations in expression levels of genes involved in fatty acid metabolism and absorption (Carrier, Deblois et al. 2004). This is consistent with the expression profile of ERRα that is predominately found in tissues and has increased capacity for fatty acid oxidation, storage and absorption (Sladek, Bader et al. 1997). The constitutive activity of ERRα is robustly stimulated by PGC-1, a co-activator that enhances fatty acid oxidation, oxidative phosphorylation and induces mitochondrial biogenesis (Schreiber, Emter et al. 2004). Small molecule antagonist against ERRα antagonized these ERRα:PGC-1 mediated responses in in vitro cellular assays but did not return them back to basal levels (Mootha, Handschin et al. 2004). These responses appear to be dependent on the presence of PPAR's and other PGC-1 transcription factor partners. Therefore ERRα augments or attenuates PPAR and PGC-1 responsive genes to external stimuli (Wende, Huss et al. 2005). ERRγ is highly expressed in metabolic active tissues during fetal development such as skeletal muscle, adipose and heart in a similar manner to ERRα (Heard, Norby et al. 2000). In the adult highest expression levels are observed in the heart, brain, kidney and pancreas (Hong, Yang et al. 1999). Its basal transcriptional activity is strongly stimulated in the presence of the PGC-1 family of transcription factors but little is known about the biological consequence of this interaction (Kamei, Ohizumi et al. 2003). In the ERRα knock-out no compensatory changes have been reported on the mRNA levels for ERRγ with the exception of in the heart where a ˜2-fold increase was shown (Huss, Torra et al. 2004). This change was correlated with an equal change to PGC-1 levels, but the extent of each PGC-1 isoform in mediating biological responses still needs to be determined. ERRγ is positively correlated with ERα positive breast cancers (Ariazi, Clark et al. 2002) and is associated with a positive prognostic outcome for anti-estrogen therapies. The later might be due that 4OHT is a potent ligand for ERRγ (Coward, Lee et al. 2001).
One of the rate limiting steps in defining biological function for orphan NR is the discovery of interacting ligands that would pharmacologically modulate its biological activity. New screening technologies have been developed for the discovery of ligands for the orphan NHR, and have assisted in the identification of ligands that can be used as tools for elucidating the biology of these receptors (Shiau, Coward et al. 2001; Rosen, Marschke et al. 2003). This approach is referred to as “reverse endocrinology” (Heyman, Mangelsdorf et al. 1992). Although a high-risk endeavor, the ERR NR are highly druggable as several reports have attested to in recent years. Pharmacological modulation of ERRα by the small molecule antagonist XCT790 has elucidated the role of ERRα in the regulation of oxidative phosphorylation genes (Busch, Stevens et al. 2004; Willy, Murray et al. 2004). X-ray crystallography studies of 4OHT and di-ethylstilbestrol (DES) showed that ERRγ and ERRγ are antagonists and the determined co-crystal structure of ERRγ provided the molecular basis of the observed antagonism (Coward, Lee et al. 2001; Greschik, Flaig et al. 2004). Phenolic acyl hydrazones have been described as ERRγ agonists although no details were given on the molecular basis for the observed agonist response (Zuercher, Gaillard et al. 2005). The present invention is directed to ligands that stabilize the ligand binding domain for all three members of the ERR family as determined using ThermoFluor® as a high throughput screening (HTS) platform (Pantoliano, Petrella et al. 2001). ThermoFluor® exploits the well characterized phenomenon of ligand induced stabilization of macromolecules (Rentzeperis, Marky et al. 1995). The technology provides competitive advantages over existing technologies since it does not require a functional response and can detect low affinity ligands (Grasberger, Lu et al. 2005; Matulis, Kranz et al. 2005).
Compounds that associate and stabilize the ligand binding domain of ERRγ have been identified. Affinities for these ligands were measured by isothermal methods and their functional response was determined by a co-activator recruitment assay. Two of the identified phenol containing ligands, BPA and ClCH3Ph, associate with potencies of 70 and 380 nM respectively, and compete for 4OHT binding, a reported antagonist of ERRγ. The structures for these two compounds were determined to resolutions of 2.1 and 2.3 Å, respectively. Superimposition of the structures with the reported constitutively active apo-form of the receptor showed no changes in the overall conformation of the receptor consistent with the ligands being functionally silent.
The Estrogen-Related Receptor 3 (ERR3), also termed Estrogen-Related Receptor gamma (ERRγ), belongs to the family of estrogen-related receptors. Although their biological function is not well understood, ERRs (ERRα, ERRβ and ERRγ) are regarded as constitutively active and no natural ligands that will regulate their function have yet been identified. Estrogen-related receptors themselves belong to the family of Nuclear Receptors (NRs). Orphan members of the nuclear receptor (NR) superfamily were initially identified by their high homology with the steroid or retinoid receptors and are hypothesized to be ligand-regulated despite the lack of a known ligand (Willy et al., 1997; Giguere, 1999)).
NRs play an important role in differentiation, development and metabolism. Their cognate ligands and relevant accessory proteins regulate highly specific biological activities. Knowledge of the ligands, accessory proteins and genes they regulate can provide new drug targets for the treatment of diseases such as diabetes, obesity, osteoporosis, heart disease and cancer.
U.S. Pat. Nos. 6,359,116 and 6,069,239 disclose the full length protein sequence of ERR3.
U.S. Application No. 20050074765 discloses a method of identifying compounds that will be useful for treatment of ERRγ and ER-mediated diseases.
U.S. Application No. 20040009558 (“the '558 application”) discloses a peptide fragment that mimicks, when fused to a polypeptide containing a DNA-binding domain, the ligand dependence of the transcriptional activity of ERRγ. The '558 application also discloses a method for selecting a compound that interacts with the Ligand Binding Pocket (LBP) of ERR3 using the relative structural coordinates according to Table 1 therein and a crystallized protein as defined therein.
Current approaches to validate the therapeutic utility of a target for the treatment of a disease rely on genomic data and annotating function by sequence analysis. Once a target is validated then chemical libraries can be selected or synthesized that are centered on known chemotypes for the particular function of the target and assayed with conventional methods. Conventional assay development is problematic for orphan targets since they rely on competitive displacement of a known ligand or rely on a functional response. With ThermoFluor®, assay development for an orphan protein is not problematic (Grasberger, Lu et al. 2005). Ligands that will interact with the protein will be identified because they will produce a positive stabilization of the protein. If the stabilizing ligand is a biochemical, then a putative function can be assigned to the orphan protein and a biochemical, functional or cell-based assay can be designed to elucidate the biology of the protein. In this invention we have demonstrated the utility of ThermoFluor® in identifying ligands that stabilize ERRγ, an orphan nuclear receptor, and used crystallography to define the molecular basis of the functionally silent response of BPA and ClCH3Ph in our cellular trans-activation and co-activator TR-FRET assays. The diversity of ligands that were found to interact with this receptor raises the possibility of the existence of a natural ligand that can regulate the activity of ERRγ. Stabilization of the receptor can result in changes of steady state levels that can impact protein levels, phosphorylation states, that can impact biological activity through cross-talk to other nuclear receptors (Kojo, Tajima et al. 2006), alter affinity for response elements (Barry and Giguere 2005) or specific co-regulator interactions (Barry, Laganiere et al. 2006). The methodology described in the identification of stabilizing ligands and the novel hydrogen bonds observed with the ligands and the ERRγ reported structures will assist in the design of selective ERRγ modulators.
There is a need to identify compounds which modulate ERRs in order to provide biologically active compounds that exert an effect on the transcriptional-activating activity of ERRs. Such molecules can modulate the response of the receptor or impact biological response of other transcription factors by competing for ancillary proteins and DNA response elements and can be useful for the treatment of metabolic and endocrine disorders.